The ability to produce skin in vitro with barrier properties equivalent to those of native epidermis remains a much sought after goal. The current proposal will utilize a number of recent improvements in the organotypic culture of skin cells to optimize the barrier properties of a highly differentiated, serum-free skin model. Medium additives, methods of culture, and the original cell source from which the epidermal cells were derived will be investigated in order to reconstruct in vivo-like skin. The modified skin tissues will be characterized in terms of histology, surface hydration, lipid content, and ultrastructure. Finally, the permeability characteristics of the in vitro tissues in relation to three model drugs spanning a range of lipophilicity will be investigated. PROPOSED COMMERCIAL APPLICATION: The improved skin tissue resulting from this research will find utilization in transdermal drug delivery studies, risk assessment related to accidental or occupational exposure of chemicals to the skin, and skin irritation studies. In addition, the methodology developed will likely improve cultured skin graft material since it has ben reported that skin tissue with improved barrier properties increases the efficacy of such grafts.